Sean M. Lynch

Cadmium alters the localization of N-cadherin, E-cadherin, and β-catenin in the proximal tubule epithelium

Recent studies on proximal tubule-derived cells in culture have shown that Cd has relatively specific damaging effects on the cadherin-dependent junctions between the cells. The objective of the present study was to determine whether Cd can affect cadherin-dependent junctions in the proximal tubule epithelium in vivo. Male Sprague-Dawley rats received subcutaneous injections of Cd (0.6 mg/kg in isotonic saline, 5 days per week for up to 6 weeks). One day each week, 24-h urine samples were collected and analyzed for protein and creatinine. After 5-6 weeks, the Cd-treated animals developed significant proteinuria, with no change in creatinine excretion. Visualization of pan-cadherin immunoreactive materials by immunoperoxidase labeling showed that Cd caused a marked reduction in the intensity of cadherin labeling associated with the apical and the basolateral surfaces of the epithelial cells of the proximal tubule, but no change in the pattern of cadherin labeling in other segments of the nephron. Results of studies utilizing specific antibodies against N-cadherin, E-cadherin, and beta-catenin showed changes in the localization of all three molecules in the proximal tubule. Assessment of cell membrane integrity with trypan blue and ethidium homodimer showed no overt evidence of death in the proximal tubule epithelial cells. Additional results showed that Cd caused only a slight increase in the total levels of glutathione and no significant peroxidation of membrane lipids, indicating only a modest level of oxidative stress. These results indicate that Cd can disrupt cadherin-dependent cell-cell junctions in the proximal tubule, and they raise the possibility that a loss of cadherin-mediated adhesion may contribute to the nephrotoxic effects of Cd.

Vitamin C protects low-density lipoprotein from homocysteine-mediated oxidation.

Homocysteine, an atherogenic amino acid, promotes iron-dependent oxidation of low-density lipoprotein (LDL). We investigated whether vitamin C, a physiological antioxidant, could protect LDL from homocysteine-mediated oxidation. LDL (0.2 mg of protein/ml) was incubated at 37 degrees C with homocysteine (1000 microM) and ferric iron (10-100 microM) in either the absence (control) or presence of vitamin C (5-250 microM). Under these conditions, vitamin C protected LDL from oxidation as evidenced by an increased lag time preceding lipid diene formation (> or = 5 vs. 2.5 h for control), decreased thiobarbituric acid-reactive substances accumulation (< or = 19 +/- 1 nmol/mg when vitamin C > or = 10 microM vs. 32 +/- 3 nmol/mg for control, p <.01), and decreased lipoprotein anodic electrophoretic mobility. Near-maximal protection was observed at vitamin C concentrations similar to those in human blood (50-100 microM); also, some protection was observed even at low concentrations (5-10 microM). This effect resulted neither from altered iron redox chemistry nor enhanced recycling of vitamin E in LDL. Instead, similar to previous reports for copper-dependent LDL oxidation, we found that vitamin C protected LDL from homocysteine-mediated oxidation through covalent lipoprotein modification involving dehydroascorbic acid. Protection of LDL from homocysteine-mediated oxidation by vitamin C may have implications for the prevention of cardiovascular disease.

Plasma thiols inhibit hemin-dependent oxidation of human low-density lipoprotein.

Oxidative modification of human low-density lipoprotein (LDL) renders it atherogenic. Previous studies demonstrated that plasma thiols promote oxidation of LDL by free ferric iron (Fe3+). The current study investigated effects of plasma thiols on oxidation of LDL by hemin, a physiological Fe3+-protoporphyrin IX complex thought to be capable of initiating LDL oxidation in vivo. In contrast to free Fe3+ which is incapable of oxidizing LDL in the absence of an exogenous reductant, hemin readily promoted LDL oxidation. During incubation of LDL (0.2 mg of protein/ml) with hemin (10 microM) at 37 degrees C for 6 h, thiobarbituric acid-reactive substances (TBARS), a marker of lipid oxidation, increased from 0.3 (+/-0.1) nmol/mg of LDL protein to a maximal concentration of 45.8 (+/-5.2) nmol/mg of LDL protein. Under the same experimental conditions, lipid-conjugated dienes, another marker of lipid oxidation, increased from non-detectable to near-maximal levels of 78-187 nmol/mg of LDL protein, and lipoprotein polyunsaturated fatty acyl-containing cholesteryl ester content decreased to 15-36% of that present in native (i.e. unoxidized) LDL. Continued incubation of LDL with hemin for up to 24 h resulted in no further significant alterations in lipoprotein levels of TBARS, lipid-conjugated dienes, and cholesteryl esters. In addition to these chemical modifications indicative of lipoprotein oxidation, agarose gel electrophoretic analysis indicated that exposure of LDL to hemin resulted in conversion of the lipoprotein to an atherogenic form as evidenced by its increased anodic electrophoretic mobility. Addition of physiological concentrations of plasma thiols (either cysteine, homocysteine or reduced glutathione; 1-100 microM, each) inhibited hemin-mediated oxidation of LDL. Thus, whereas the maximal TBARS concentration was achieved following 6 h of incubation of LDL with hemin alone, addition of thiol extended the time required to attain maximal TBARS concentration to > or = 12 h. Similar antioxidant effects of thiols on formation of lipid-conjugated dienes, loss of cholesteryl esters, and lipoprotein anodic electrophoretic mobility were also observed. However, all thiols were not equally effective at inhibiting hemin-dependent LDL oxidation. Thus, whereas reduced glutathione was most effective at inhibiting hemin-dependent LDL oxidation, an intermediate effect was observed for homocysteine, and cysteine was least effective. The inhibition of hemin-mediated LDL oxidation by plasma thiols reported here confirms a previous observation that, under certain conditions, thiols can function as antioxidants, but contrasts with the previously documented pro-oxidant effect of the same thiols on oxidation of LDL by free Fe3+. These contrasting effects of plasma thiols on hemin- and free Fe3+-mediated LDL oxidation indicate that, in vivo, the ability of thiols to function as either anti- or pro-oxidants during LDL oxidation may, at least in part, be determined by the type of oxidant stress to which the lipoprotein is exposed.

Interprofessional workshop to improve mutual understanding between pharmacy and medical students.

Objective. To measure changes in pharmacy and medical students’ physician-pharmacist collaboration scores resulting from a workshop designed to promote understanding of the others’ roles in health care. Methods. More than 88% of first-year pharmacy (n = 215) and medical (n = 205) students completed the Scale of Attitudes Toward Physician-Pharmacist Collaboration on 3 occasions in order to establish a baseline of median scores and to determine whether the scores were influenced by an interprofessional workshop. Results. Participation in the interprofessional workshop increased pharmacy students’ collaboration scores above baseline (p=0.02) and raised the scores of medical students on the education component of the collaboration survey instrument (p=0.015). The collaboration scores of pharmacy students greatly exceeded those of medical students (p<0.0001). Conclusion. A workshop designed to foster interprofessional understanding between pharmacy and medical students raised the physician-pharmacist collaboration scores of both. Crucial practical goals for the future include raising the collaboration scores of medical students to those of pharmacy students.

Differential modulation of microglia superoxide anion and thromboxane B2 generation by the marine manzamines

BackgroundThromboxane B2 (TXB2) and superoxide anion (O2-) are neuroinflammatory mediators that appear to be involved in the pathogenesis of several neurodegenerative diseases. Because activated-microglia are the main source of TXB2 and O2- in these disorders, modulation of their synthesis has been hypothesized as a potential therapeutic approach for neuroinflammatory disorders. Marine natural products have become a source of novel agents that modulate eicosanoids and O2- generation from activated murine and human leukocytes. With the exception of manzamine C, all other manzamines tested are characterized by a complex pentacyclic diamine linked to C-1 of the β-carboline moiety. These marine-derived alkaloids have been reported to possess a diverse range of bioactivities including anticancer, immunostimulatory, insecticidal, antibacterial, antimalarial and antituberculosis activities. The purpose of this investigation was to conduct a structure-activity relationship study with manzamines (MZ) A, B, C, D, E and F on agonist-stimulated release of TXB2 and O2- from E. coli LPS-activated rat neonatal microglia in vitro.ResultsThe manzamines differentially attenuated PMA (phorbol 12-myristate 13-acetate)-stimulated TXB2 generation in the following order of decreasing potency: MZA (IC50 <0.016 μM) >MZD (IC50 = 0.23 μM) >MZB (IC50 = 1.6 μM) >MZC (IC50 = 2.98 μM) >MZE and F (IC50 >10 μM). In contrast, there was less effect on OPZ (opsonized zymosan)-stimulated TXB2 generation: MZB (IC50 = 1.44 μM) >MZA (IC50 = 3.16 μM) >MZC (IC50 = 3.34 μM) >MZD, MZE and MZF (IC50 >10 μM). Similarly, PMA-stimulated O2- generation was affected differentially as follows: MZD (apparent IC50<0.1 μM) >MZA (IC50 = 0.1 μM) >MZB (IC50 = 3.16 μM) >MZC (IC50 = 3.43 μM) >MZE and MZF (IC50 >10 μM). In contrast, OPZ-stimulated O2- generation was minimally affected: MZB (IC50 = 4.17 μM) >MZC (IC50 = 9.3 μM) >MZA, MZD, MZE and MZF (IC50 > 10 μM). From the structure-activity relationship perspective, contributing factors to the observed differential bioactivity on TXB2 and O2- generation are the solubility or ionic forms of MZA and D as well as changes such as saturation or oxidation of the β carboline or 8-membered amine ring. In contrast, the fused 13-membered macrocyclic and isoquinoline ring system, and any substitutions in these rings would not appear to be factors contributing to bioactivity.ConclusionTo our knowledge, this is the first experimental study that demonstrates that MZA, at in vitro concentrations that are non toxic to E. coli LPS-activated rat neonatal microglia, potently modulates PMA-stimulated TXB2 and O2- generation. MZA may thus be a lead candidate for the development of novel therapeutic agents for the modulation of TXB2 and O2- release in neuroinflammatory diseases. Marine natural products provide a novel and rich source of chemical diversity that can contribute to the design and development of new and potentially useful anti-inflammatory agents to treat neurodegenerative diseases.

Critical Thinking and Reflection Exercises in a Biochemistry Course to Improve Prospective Health Professions Students’ Attitudes Toward Physician-Pharmacist Collaboration

Objective. To determine the impact of performing critical-thinking and reflection assignments within interdisciplinary learning teams in a biochemistry course on pharmacy students’ and prospective health professions students’ collaboration scores. Design. Pharmacy students and prospective medical, dental, and other health professions students enrolled in a sequence of 2 required biochemistry courses. They were randomly assigned to interdisciplinary learning teams in which they were required to complete case assignments, thinking and reflection exercises, and a team service-learning project. Assessment. Students were asked to complete the Scale of Attitudes Toward Physician-Pharmacist Collaboration prior to the first course, following the first course, and following the second course. The physician-pharmacist collaboration scores of prospective health professions students increased significantly (p<0.001). Conclusions. Having prospective health professions students work in teams with pharmacy students to think and reflect in and outside the classroom improves their attitudes toward physician-pharmacist collaboration.

Vitamin C attenuates hypochlorite-mediated loss of paraoxonase-1 activity from human plasma

Paraoxonase 1 (PON1) is a cardioprotective enzyme associated with high-density lipoprotein (HDL). We tested the hypothesis that vitamin C protects HDL and PON1 from deleterious effects of hypochlorous acid, a proinflammatory oxidant. In our experiments, HDL (from human plasma) or diluted human plasma was incubated with hypochlorite in either the absence (control) or presence of vitamin C before measuring chemical modification and PON1 activities. Vitamin C minimized chemical modification of HDL, as assessed by lysine modification and accumulation of chloramines. In the absence of vitamin C, chloramines accumulated to 114 +/- 4 micromol/L in HDL incubated with a 200-fold molar excess of hypochlorite; but addition of vitamin C (200 micromol/L) limited formation to 36 +/- 6 micromol/L (P < .001). In plasma exposed to hypochlorite, IC(50) values of 1.2 +/- 0.1, 9.5 +/- 1.0, and 5.0 +/- 0.6 mmol/L were determined for PON1s phosphotriesterase, arylesterase, and (physiologic) lactonase activities, respectively. Vitamin C lessened this inhibitory effect of hypochlorite on PON1 activities. In plasma supplemented with vitamin C (400 micromol/L), PON1 phosphotriesterase activity was 72% +/- 17% of normal after incubation with hypochlorite (2 mmol/L), compared with 42% +/- 6% for unsupplemented plasma (P < .05). Similar effects were seen for other PON1 activities. In some experiments, vitamin C also appeared to reverse hypochlorite-mediated loss of PON1 phosphotriesterase activity; but this effect was not observed for the other PON1 activities. In conclusion, vitamin C attenuated hypochlorite-mediated loss of PON1 activity in vitro and may, therefore, preserve cardioprotective properties of HDL during inflammation.

Challenging Medical Students to Confront their Biases: A Case Study Simulation Approach

We used three approaches to determine whether first-year medical students would begin to confront their biases in response to a simulated encounter with an incarcerated, African-American patient. The patient presented with fatigue in a Biochemistry course workshop. Two hundred five students watched and helped a classmate conduct a simulated interview with the patient who had been imprisoned for attempted murder. We then studied whether the students confronted their biases against the patient using (a) a survey of individual students regarding these biases, (b) one of a number of questions on a formal assignment concerning the case completed in a team format, and (c) an unprompted extra-credit opportunity to reflect as a team on issues of their choice. On the survey, eighty five percent of students confronted their biases against the patient, and they began to reflect critically about these biases. Critical reflection on teams occurred more frequently outside the formally assigned exercise (Effect Size = 0.75, crucial practical importance). Thus, most first-year medical students can be led, even in basic sciences courses, to confront their biases. In this way, they may also begin to mitigate their biases against patients. Such self-regulation of biases by health care professionals on a regular basis should help to decrease health care disparities.

Does Critical Reflection by Biochemistry Learning Teams Foster Patient-centered Beliefs among Medical Students?

The authors measured patient-centered orientation scores of first-year osteopathic medical students before vs. after implementing exercises to foster critical reflection by learning teams. In the 2008–09 academic year, 60 of a total of 130 classroom hours were devoted to team-based learning (TBL) in a sequence of three Biochemistry courses (1500, 1501 and 1502). TBL was modified to include required and extra-credit team critical reflection assignments. Index questions were used to compare student learning on their own using modified TBL to learning facilitated by instructors in the preceding year. Individual student performances on index questions improved using modified TBL as the sequence of three courses progressed from 8% lower than in the prior year for the first course (p<0.001) to 7% higher than in the prior year for the third course (p<0.005). Moreover, team members cooperated more on team tests as they progressed from Biochemistry 1500 through Biochemistry 1502 (p<0.01). Finally, students became more patient-centered and caring (p<0.0001) using modified TBL, whereas the reverse was observed (p<0.01) prior to implementing exercises to foster critical reflection by team-based learning teams. Thus, TBL, modified to include opportunities for team critical reflection, fostered both cooperation and patient-centered orientations in first-year medical students.

Inclusion of calcium during isolation of high-density lipoprotein from plasma maintains antioxidant function.

We investigated how inclusion of calcium during isolation of high-density lipoprotein (HDL) affected its antioxidant function. Following isolation, HDL was dialyzed against 0.154 M NaCl without or with added calcium (1mM). HDLs paraoxonase 1 activity was unaffected by calcium treatment (87 ± 11% of normal vs. 89 ± 16% of normal, P=0.826). In contrast, whereas HDL dialyzed with calcium inhibited oxidation of low-density lipoprotein (LDL) by 87 ± 10%, HDL dialyzed without calcium inhibited oxidation by only 58 ± 19% (P=0.004). Thus, inclusion of calcium during isolation is important for maintaining HDLs antioxidant function.